Abstract
BackgroundControlled release of proteins bound to conventional bone substitutes is still insufficient. Therefore, this study evaluates in-vitro release kinetics of the model protein FITC-BSA (fluorescein conjugated bovine serum albumine) from insoluble bovine collagenous bone matrices (ICBM) with different polymer coatings. Analyzes aim at comparing FITC-BSA release from uncoated versus coated ICBM over time to find bone substitute coatings with consistent release profiles.MethodsRelease kinetics of FITC-BSA from uncoated as well as coated ICBM with five different polymers (RESOMER R 203 H, RG 503 H, RG 504 H, RG 505, L 206 S) were measured over a period of 11 days (d). Measurements were conducted after 6 h (h), 12 h, 24 h, 3 d, 5 d, 7 d, 9 d and 11 d with six samples for each coated ICBM. Two groups were formed (1) with and (2) without medium change at times of measurement. For each group ANOVA with post-hoc Bonferroni testing was used. Scanning electron microscopy assessed morphologic differences between ICBM coating.ResultsIn group 1 approx. 70% of FITC-BSA release from uncoated ICBM occurred after 6 h compared to approx. 50% in group 2. Only polymers with medium inherent viscosity, i.e. RESOMER RG 503 H, constantly showed significantly more FITC-BSA release throughout 11 d than uncoated ICBM (p = 0.007). The same was found for group 2 (p = 0.005). No significant differences between PLA and PLGA polymers were found. Scanning electron microscopy results indicate a weak adhesion of polymer coatings to ICBM explaining its rather weak retentive effect on overall FITC-BSA release.ConclusionsMedium molecular size polymers reduce the overall released FITC-BSA from ICBM over time. In clinical practice these polymers may prove ideal for bone substitute materials.
Highlights
Controlled release of proteins bound to conventional bone substitutes is still insufficient
Since the release kinetics of substances from polymer-coated insoluble bovine collagenous bone matrices (ICBM) depend on the physicochemical properties of the polymers, polylactic acid (PLA) and PGLA were designed with specific grades of hydrophilic, crystalline and degradation properties
Determination of the standard curve for fluorescence measurement of FITC-BSA Preliminary tests showed a linear correlation between concentrations of FITC-BSA and the measured fluorescence values detected by GENios© (Pearson: r = 0.993, p = 0.000)
Summary
Controlled release of proteins bound to conventional bone substitutes is still insufficient. This study evaluates in-vitro release kinetics of the model protein FITC-BSA (fluorescein conjugated bovine serum albumine) from insoluble bovine collagenous bone matrices (ICBM) with different polymer coatings. Modern tissue engineering of bone replacement materials aims at combining new carrier matrices with cells and cytokines (i.e. interleukin-1 [IL-1], interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) to best possibly mimic autologous bone [3]. Recombinant bone morphogenetic protein-2 (rhBMP-2), a strong initiating protein for bone regeneration, was combined with ICBM and approved by the American Food and Drug Administration (FDA) for medical treatment of vertebral bone defects (InFuse®, Medtronic, Memphis, Tennessee, USA) [11, 12] Observed side effects such as inflammation, excessive bone formation and questionable carcinogenic potency were attributed to the uncontrolled release of cytokines and proteins from ICBM [13]. Due to the linear relationship between fluorescence and concentration fluorescein isothiocyanate-labeled bovine serum albumin (FITCBSA) is often used as a model protein for pharmacological release kinetics testing [18, 19]
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